Method for detecting cations in a test sample utilizing chromogenic cryptahemispherands

ABSTRACT

The present invention resides in the discovery of a class of compounds defined herein as &#34;chromogenic cryptahemispherands&#34; useful for the measurement of ions, in particular, ions in aqueous solution, which have the structure ##STR1## wherein: R, same or different, is hydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl; 
     R&#39;, same or different, is lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl; 
     R&#34;, same or different, is hydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl; 
     Q is a chromogenic moiety capable of providing the appearance of or change in color, or which is otherwise capable of providing a detectable response in the presence of a particular cation; 
     a, b, m and n, same or different, are 1 to about 3; and x, y, same or different, are 1 to about 4. 
     A test device utilizing one or more of the compounds for performing such measurements is also disclosed.

CONTENTS Section

1. Introduction

2. Background of the Invention

2.1 Ion-selective Electrodes

2.2 Liquid/Liquid Partitioning

2.3 Fluorescent Anions

2.4 Reporter Substances

2.5 Ionophores

2.5.1 Podands

2.5.2 Corands

2.5.3 Cryptands

2. 5.4 Hemispherands

2.5.5 Spherands

2.5.6 Cryptahemispherands

2.6 Chromogenic Ionophores

2.7 Synopsis

3. Brief Description of the Drawings

4. Summary of the Invention

Definitions

5.1 "Ionophore"

5.2 "Chromogenic"

5.3 "Detectable Response"

5.4 "Lower Alkyl, Lower Alkylidene, Lower Alkenyl"

5.5 "Aryl"

5.6 "Electron Withdrawing Group"

6. The Chromogenic Cryptahemispherand

6.1 Cationic Adaptability

6.2 The Chromogenic Moiety

6.3 Presently Preferred Embodiment

7. The Test Composition

Section

8. The Test Device

8.1 The Carrier Matrix

8.2 Making the Test Device

9. Use of the Invention

10. Experimental

10. 1 Synthesis of a Preferred Chromogenic Cryptahemispherand

10. 2 A Preferred Aqueous System for Potassium Determination

10. 3 Use of a Preferred Aqueous System for Serum Determination ofPotassium

10.4 Effect of pH on Potassium/Sodium Selectivity in a Liquid/LiquidPartitioning System

10. 5 Effect of pH and a Water-Miscible Organic Solvent onPotassium/Sodium Selectivity in an Aqueous System

10. 6 A Model Test Device

10. 7 Test Device for Detecting Potassium in Serum

10. 8 A Preferred Aqueous System for Sodium (Rate) Measurement

10.9 A Preferred Aqueous System for Sodium (End Point) Measurement

10.10 Test Device for Detecting Sodium Ions

11. What is Claimed

12. Abstract of the Disclosure

1. INTRODUCTION

The present invention relates to a novel class of compounds useful forthe measurement of ions, in particular ions in aqueous solution, and toa test means or device utilizing one or more of the compounds forperforming such measurements. The invention provides a quick, facile wayof assaying such ions whereby results are available to the assayistmomentarily after merely contacting a test sample solution with the testmeans or device. There is no need for cumbersome, expensive electronicequipment such as ion-selective electrodes, flame photometers, atomicabsorption spectrophotometers or the like. Nor is it necessary to resortto time-consuming wet chemistry techniques such as titration and otherlaboratory procedures The present invention enables the analyst tomerely contact the test sample with a test composition or a dry testdevice, test slide, or similar test means or configuration, and observeany color change or other detectable response. Finally, the presentinvention enables an unusually fast assay and unexpectedly high degreeof selectivity, thereby permitting the detection of relatively lowconcentrations of an analyte ion even in solutions having relativelyhigh concentrations of different, potentially interfering ions, whileproviding selectivity and accuracy to a degree heretofore unknown.

The determination of aqueous ion concentration has application innumerous technologies. In the water purification art, calciumconcentration must be carefully monitored to assess the degree ofsaturation of an ion exchange resin deionizer. Measurement of sodium andother ions in seawater is important in the preparation of drinking wateraboard a ship at sea. Measurement of the potassium level in blood aidsthe physician in the diagnosis of conditions leading to muscleirritability and excitatory changes in myocardial function. Suchconditions include oliguria, anuria, urinary obstruction and renalfailure due to shock.

Needless to say, a rapid, easy-to-perform method for determining thepresence and concentration of a specific ion in aqueous samples wouldgreatly enhance the state of these technologies, as well as any otherswhere such quick, accurate determinations would be beneficial. Thus, forexample, if a medical laboratory technician could accurately measure thepotassium or sodium level of a serum, whole blood, plasma or urinesample in a matter of seconds or minutes, it would aid the physician inearly diagnosis, and laboratory efficiency would increase manyfold. Thepresent invention affords these and other unexpected advantages.

2. BACKGROUND OF THE INVENTION

Prior to the present invention, methods for determining ions in solutionincluded flame photometry, atomic absorption photometry, ion-selectiveelectrodes, multiple liquid phase partitioning and colorimetric slides.The use of certain compounds and compositions which selectively complexwith, and therefore isolate, certain ions from the sample solution hasbecome popular in ion-selective electrodes. These substances, known asionophores, have the capability of selectively isolating ions from theircounterions and other ions in a test sample, thereby causing a chargeseparation and a corresponding change in electrical conductivity in thephase containing the ionophore. Illustrative of other uses of theion/ionophore phenomenon include ion assays utilizing membraneelectrodes, liquid/liquid partitioning, fluorescence, various reportersubstances, and chromogenic derivatives of certain ionophoric compounds.

2.1 Ion-Selective Electrodes (ISE) When two solutions having differentconcentrations of ions are separated by an electrically conductivemembrane, an electromotive force (EMF) can be generated. The EMFdeveloped by such a system is a function of concentration or ionicactivity of the solutions on either side of the membrane. Thisphenomenon is expressed mathematically by the well-known Nernst Equation##EQU1## in which E is the EMF of the particular system, F is theFaraday Constant, R is the gas constant, T is the temperature in °K andγ and c are, respectively, the activity coefficients and molalconcentrations of the ion under study. The subscript 1 designates thesolution on one side of the membrane; the subscript 2 denotes thesolution on the other side. The charge of the ion involved in thereaction is denoted by n.

In such membrane separation cells, the membrane can be a simple frittedglass barrier, allowing a small but measurable degree of ion diffusionfrom one solution to the other. Alternatively, a nonporous, electricallynonconductive film, such as polyvinyl chloride, impregnated with anionophore can be employed. In the absence of the ionophore the film isan insulator and no EMF can be measured; when blended with an ionophore,charged ions are bound to the film and a small, measurable current canbe induced to flow. Because the ionophore is selective in its affinity,and thus will bind only certain specific ions, such cells are ionselective. Any measurable EMF is due solely to the presence of thoseions.

It is known that certain antibiotics, such as valinomycin, have aneffect on the electrical properties of phospholipid bilayer membranes(biological membranes), such that these antibiotics effectsolubilization of cations within the membrane, in the form of mobilecharged complexes, thereby providing a "carrier" mechanism by whichcations can cross the insulating hydrophobic or hydrocarbon interior ofthe membrane. Such complexes have the sole purpose of carrying thecharge of the complex through the membrane. In an ISE they cause avoltage differential which can be determined between solutions on eitherside of the ISE membrane.

Thus, a cell for determining potassium ion can be produced through useof an ionophore specific for potassium (K⁺), e.g. valinomycin. In thepresence of K⁺, valinomycin produces a concentration gradient across amembrane by binding and transporting the ion, thus generating apotential across the membrane. A reference concentration of K⁺ is placedon one side of the membrane and the test sample on the other. The EMFdeveloped is measured using external reference electrodes and used tocalculate the unknown concentration from equation (1). Because only K⁺binds to the valinomycin in the membrane, the conductive path onlyappears for K⁺. Therefore, the EMF developed is attributable solely tothe K⁺ concentration gradient across the membrane.

The current flowing across the membrane is so small that no significantquantity of K+ or counterion is transported through it. Electricalneutrality of the membrane is maintained either by a reverse flow ofhydrogen ions (protons), or by a parallel flow of OH--.

A major difficulty in the use of such ion-selective electrodes has beenthe marked reduction of accuracy, selectivity and speed of response overtime. Further, small changes in ion concentration produce such smallchanges in EMF that sophisticated voltmeter equipment is required.

Swiss patent application Ser. No. 11428/66, filed Aug. 9, 1966,describes the use of porous membranes impregnated with macrocyclicderivatives of amino and oxy-acids in ion-sensitive electrodes.Materials used to form the membrane are glass frits and other porousmembranes. Such electrodes are said to be effective in measuring ionactivities.

U.S. Pat. No. 4,053,381, issued to Hamblen, et al., discloses similartechnology, and utilizes an ion specific membrane having ion mobilityacross it.

Liquid/Liquid Partitioning

Another known application of ionophores in ion determination is throughliquid/liquid partitioning. Eisenman et al., J. Membrane Biol., 1,294-45 (1969), disclose the selective extraction of cations from aqueoussolutions into organic solvents via macrotetralide actin antibiotics. Inthis procedure, a hydrophobic ionophore is dissolved in an organicsolvent immiscible with water. The technique involves shaking an organicsolvent phase containing the antibiotics with aqueous solutionscontaining cationic salts of lipid-soluble colored anions, such aspicrates and dinitrophenolates. The intensity of color of the organicphase is then measured spectrophotometrically to indicate how much salthas been extracted. Phase transfer has also been studied by Dix et al.,Angew, Chem. Int. Ed. Engl., 17, 857 (1978) and is reported in reviewsincluding Burgermeister et al., Top. Curr. Chem., 69, 91 (1977); Yu etal., "Membrane Active Complexones," Elsevier, Amsterdam (1974); andDuncan, "Calcium in Biological Systems,"Cambridge University Press(1976).

Sumiyoshi, et al., Talanta, 24, 763-765 (1977) describe another methoduseful for determining K+ in serum. In this technique serum isdeproteinated by trichloroacetic acid, an indicator dye is added, andthe mixture shaken with a solvent such as chloroform containingvalinomycin.

Partitioning of a compound is rapid and effective between liquids, asshown by Eisenman, because of the mobility of the ionophore carrier andions in their respective phases, which allows the transported species todiffuse rapidly away from the interface. Such a mechanism is normallyimpossible in the solid phase, because of the rigidity, immobility andessentially zero diffusion of materials in a solid phase.

2.3 Fluorescent Anions

Yet another approach to the measurement of ion activity in aqueoussolutions utilizes fluorescent anions. Feinstein, et al., Proc. Nat.Acad. Sci. U.S.A., 68, 2037-2041 (1971). It is stated that the presenceof cation/ionophore complexes in organic solvents is known, but thatcomplex formation in purely aqueous media had theretofore not beendetected. Feinstein, et al., demonstrated the existence of suchcomplexes in water through the use of the fluorescent salts1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl sulfonate.

It was found that interaction of the ionophore/cation complex with thefluorescent dyes produced enhanced fluorescence emission, increasedlifetime and polarization, and significant blue-shift at the emissionmaxima of the fluorescence spectra. At constant concentrations ofionophore and fluorophore, the intensity of fluorescence emission wasfound to be a function of cation concentration.

2.4 Reporter Substances

As indicated supra, anionic dyes and fluorescers can be induced to enterthe organic phase of a two-phase liquid system by the presence in thatphase of a cation/ionophore complex. Thus these detectable anions can besaid to "report" the presence of the cation trapped by the ionophore inthe organic phase.

Other reporter substances which are not ionic in nature can be inducedby the ionophore/cation complex to undergo a reaction yielding adetectable product. An example is the reaction sequence reported in U.S.Pat. No. 4,540,520 whereby a cation/ionophore complex induces a phenolto become deprotonated, thus initiating a coupling reaction to form acolored product. The so-called Gibbs Reaction is typical of such areporter substance-producing reaction, in which2,5-cyclohexadiene-1-one-2,6-dichloro-4-chloroimine couples with adeprotonated phenol to form a colored product and HCl.

2.5 Ionophores

The term "ionophore" embraces many diverse molecules, all of which arerelated by their unique capacity to bind with certain charged species tothe relative exclusion of others, and which do so in a fashion which, atleast to some degree, enables the ionophore molecule to electricallyshield the ion from its environment. Indicative of this phenomenon isthe liquid/liquid partitioning technique described above. The ionophore,because of its unique structure and its multitude of electron rich orelectron deficient atoms ("donor atoms" or "receptor atoms",respectively) enables an ion such as sodium or potassium to enter anonpolar organic phase.

Ionophores include naturally occurring compounds, such as valinomycin,as well as compounds of the structural categories of podands, corands,cryptands, hemispherands, spherands and cryptahemispherands.

2.5.1 Podands

Ions can be selectively complexed with certain acyclic compounds. Forexample, a linear chain which contains a regular sequence of electronrich donor atoms, such as oxygen, sulfur or nitrogen, has the capabilityof associating with positively charged ions to form complexes. The mainstructural difference between podands and other ionophores is theopenness or acyclic nature of their structures. Thus, podands can besubcategorized into monopodands, dipodands, tripodands, etc. Amonopodand, therefore, is a single organic chain containing donor orreceptor atoms, a dipodand is two such chains connected to a centralmoiety capable of variable spacial orientation, and a tripodand is threechains attached to a central moiety.

2.5.2 Corands

The corands are monocyclic compounds which contain electron donor atomsor acceptor atoms, which are electron rich or deficient, and which arecapable of complexing with particular cations or anions because of theirunique structures. Included in this term are the crown ethers in whichthe monocyclic ring contains oxygen as the donor atoms. Other corandsare compounds which contain an assortment of electron rich atoms such asoxygen, sulfur and nitrogen. Because of the unique sizes and geometricsof particular corands, they are adaptable to complexing with variousions. In so complexing, the electron rich atoms, such as the oxygens ina crown ether, become spacially oriented towards the electron deficientcation. The carbon atom segments of the chain are simultaneouslyprojected in a direction outwards from the ion. Thus, the resultantcomplex is charged in the center but is relatively hydrophobic at itsperimeter.

2.5.3 Cryptands

The cryptands are the polycyclic analogs of the corands. Accordingly,they include bicyclic and tricyclic multidentate compounds. In thecryptands, the cyclic arrangement of donor atoms is three dimensional inspace, as opposed to the substantially planar configuration of thecorand. A cryptand is capable of virtually enveloping the ion in threedimensional fashion and, hence, is capable of strong bonds to the ion informing the complex. As with the corands, the donor atoms can includesuch atoms as oxygen, nitrogen and sulfur.

2.5.4 Hemispherands

Hemispherands are macrocyclic or macropolycyclic ionophore systems, suchas cryptands, whose cavities are partially preorganized for binding bythe rigidity of the hydrocarbon support structure and the spatial andorientational dictates of appended groups.

2.5.5 Spherands

Spherands are macrocyclic or macropolycyclic ionophore systems whosecavities are fully preorganized by their synthesis, as opposed tobecoming organized during complexing such as with an ion.

2.5.6 Cryptahemispherands

Cryptahemispherands combine the partially preorganized cavity featuresof the hemispherand, but contain multiple other ligand-gatheringfeatures of the cryptands.

2.6 Chromogenic Ionophores

Certain compounds have been studied which are capable not only ofbehaving as ionophores by forming cation complexes but which, when socomplexed, exhibit a detectable formation of or change in color. Thus,experiments were published in 1977 whereby chromophoric moieties werecovalently attached to ionophores to achieve a color response topotassium (Tagaki, et al., Analytical Letters, 10 (13), pp. 1115-1122(1977)). There it is taught to couple covalently a chromophoric moietysuch as 4-pigryl-amino- to an ionophore such as benzo-15-crown-5.Moreover, U.S. Pat. No. 4,367,072 mentions many crown ethers, cryptandsand podands covalently substituted with a chromophoric group, such as##STR2## Yet another reference, German Offenlegungschrift 32 02 779,published Aug. 4, 1983 discloses a chromogenic cryptand structure.

2.7 Synopsis

Many technological developments have occurred since the earlyrecognition that antibiotics such as valinomycin are capable ofcomplexing certain ions and transporting them into the hydrophobicinternal region of a cell membrane and, ultimately, into the cellnucleus. This basic ionophore discovery has led to the invention of amyriad of assay techniques for such ions as potassium, sodium, calciumand others; and has spawned a variety of diagnostic procedures ofinvaluable assistance to the chemist and physician. Moreover, countlessnew ionophore compounds have been discovered and invented of suchchemical and structural diversity and complexity as to engender a wholenew area of organic chemistry.

Certain applications of these technologies to ion determination,however, have met with problems. Although ionophores can possess highion selectivity, the presence of high concentrations of other ionsrelative to the ion of interest can lead to interference in the desiredresult. Thus, if an ionophore were to have a specificity ratio of 50:1for complexing with ion X+ over ion Y+, nevertheless if Y⁺ were presentin solution at a concentration 50 times that of X⁺ the resultantselectivity of the system for X⁺ would be diminished to such a greatextent as to render the ionophore practically useless as an assayreagent for X⁺. Such disparity of concentrations occurs, for example, inblood where normal sodium/potassium concentration ratios are in theneighborhood of 35:1.

Moreover, some prior art assays utilizing prior art ionophores haveheretofore required a highly alkaline medium in order to functionusefully, and aspects which contribute to poor shelf life as well ascorrosiveness. Such systems also require a hydrophobic phase to containor segregate the ionophore from the aqueous test sample, thus leading toorganic/aqueous systems which respond relatively slowly.

Thus, it would be desirable to greatly increase selectivity in achromogenic ionophore, thereby overcoming interference from competingions present at much higher concentrations. Likewise, it would bedesirable to obviate the need for harshly alkaline conditions and amultiphasic system. These and other unexpected advantages have beenrealized through utilizing the unique compounds described herein.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The appended drawings are presented to further describe the invention,and to assist in its understanding through clarification of its variousaspects.

FIGS. 1A, 1B and 1C describe a reaction pathway for synthesizing apreferred chromogenic cryptahemispherand discussed in Section 6.3 hereinand shown in FIG. 5.

FIG. 2 portrays the linear dose/response curve obtained from thepreferred embodiment of the invention described in Section 10.2 herein.

FIG. 3 shows the comparative data between the method of the presentinvention and the standard ISE method for potassium assay in randomserum samples as described in Section 10.3 herein.

FIG. 4 provides a dose/response curve for various potassium levelsutilizing the test device of the present invention described in Section10.5 herein.

FIG. 5 depicts the structure of a preferred embodiment of the presentinvention whereby the compound shown is selective for potassium ionassay.

FIG. 6 depicts the structure of a preferred embodiment of the presentinvention whereby the compound shown is selective for sodium ion in arate measurement.

FIG. 7 depicts the structure of a preferred embodiment of the presentinvention whereby the compound shown is selective for sodium ion in anend point determination.

4. SUMMARY OF THE INVENTION

Briefly stated, the present invention resides in the discovery of a newclass of compounds defined herein as "chromogenic cryptahemispherands",which have the structure (I): ##STR3## wherein: R, same or different, ishydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl;

R', same or different, is lower alkyl, lower alkylidene, lower alkenyl,allyl or aryl;

R", same or different, is hydrogen, lower alkyl, lower alkylidene, loweralkenyl, allyl or aryl;

Q is a chromogenic moiety capable of providing the appearance of orchange in color, or which is otherwise capable of providing a detectableresponse in the presence of a particular cation;

a, b, m and n, same or different, are 1 to about 3; and

x, y, same or different, are 1 to about 4.

This discovery led to further discoveries, including a composition fordetecting the presence of an ion in solution, such as potassium andsodium, and a method for its use. The composition comprises the compoundand a buffer capable of providing a pH in the range of about 5-9.Incorporation of the composition into a carrier matrix provides a drytest device for use in determining specific ions in solution. Both thecomposition and the device are utilized by contacting either with a testsample suspected of containing the ion of interest, and observing adetectable response.

Finally, the process for making the compounds of the present inventionis a further part of the present invention, entailing truly innovativeorganic synthesis, and which enabled the synthesis of a preferredembodiment of the unique compounds of the present invention. Thepreferred process comprises a synthesis sequence such as is described inFIGS. 1A, 1B and 1C.

The scope of the invention, including the compound, composition and testdevice; and their use, synthesis and preparation, and experimentalresults are set forth in Sections 4-10 herein, and in the appendedclaims.

5. DEFINITIONS

Certain terms used in the present discussion should at this point bementioned to assure that the reader is of the same mind as the authorsas to their respective meanings. Thus the following definitions areprovided to clarify the scope of the present invention, and to enableits formulation and use.

5.1 Ionophore

The term "ionophore" includes, broadly, molecules capable of forming acomplex with an ion in solution. For example the cyclic polypeptide,valinomycin, binds selectively to potassium ions in solution to form acationic complex. Also included in the term are crown ethers, cryptands,podands, spherands, hemispherands and cryptahemispherands.

5.2 Chromogenic

As used herein, "chromogenic" is intended as meaning that characteristicof a chemical system whereby a detectable response is generated inresponse to an external stimulus. Thus, for example, acryptahemispherand is chromogenic where it is capable of exhibiting adetectable response upon complexing with an ion, which detectableresponse is not limited solely to change in color as defined below.

5.3 Detectable Response

By the term "detectable response" is meant a change in or appearance ofa property in a system which is capable of being perceived, either bydirect observation or instrumentally, and which is a function of thepresence of a specific ion in an aqueous test sample. Some examples ofdetectable responses are the change in or appearance of color,fluorescence, phosphorescence, reflectance, chemiluminescence, orinfrared spectrum which are referred to generally as chomogenicresponses. Other examples of detectable responses may be the change inelectrochemical properties, pH and nuclear magnetic resonance.

5.4 Lower Alkyl, Lower Alkylidene, Lower Alkenyl

The term "lower alkyl", as used in the present disclosure includes analkyl moiety, substituted or unsubstituted, containing about 1-4 carbonatoms. Included in the meaning of lower alkyl are methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl. These may beunsubstituted, or they may be substituted provided any such substituentsdo not interfere with the operation or functioning of the presentlyclaimed test means or device in its capability to detect ions. "Loweralkylidene" is used in the same context as "lower alkyl", but designatesan alkylene or alkylidine group (i.e., a divalent alkyl) having 1-4carbon atoms. Thus, lower alkylidene includes methylene, ethylidene,n-propylidene, isopropylidene, n-butylidene, sec-butylidene andtert-butylidene. "Lower alkenyl" means vinyl or lower alkyl substitutedvinyl.

Substituent groups can be selected with a wide degree of latitude,although in general they are chosen to accommodate the intended use ofthe ionophore of the present invention in complexing with a particularcation. Thus in the case where the cryptahemispherand is designed tocomplex with a cation such as potassium, the substituent is usuallyelectrically neutral, such as hydrogen or methyl.

5.5 Aryl

By the term "aryl" is meant groups having one or more six-memberedaromatic ring systems. Such ring systems can be heterocyclic, such aspyridinyl (NC₅ H₄ --), or can be homocyclic, such as phenyl (C₆ H₅ --),benzyl (C₆ H₅ CH₂ --) and naphthyl. Aryl groups can be substituted orunsubstituted, provided that in the former case the substituent notinterfere with the intended utility of the invention, i.e., thedetection of ions in solution.

As in the case of substituent groups for lower alkyl and alkylidene, awide latitude of substitution obtains for aryl groups, depending on theuse of the ultimate chromogenic cryptahemispherand.

5.6 Electron Withdrawing Group

By the term "electron withdrawing group" is meant substituent groupssuch as NO₂, CF₃, CN, COOR.

6. THE CHROMOGENIC CRYPTAHEMISPHERAND

The chromogenic cryptahemispherand of the present invention, genericallydepicted as compound (I) in Section 4, supra, allows a significantdegree of latitude as to its geometry and chemical nature, dependentupon selection of the variable parameters such as R, R', R", Q, a, b, mand n, x and y. It is careful selection of these parameters that permitstailoring of the molecule to alter ion selectivity. Thus by followingthe teachings herein, molecules can be custom synthesized such that theinternal cavity of the bicyclic structure can vary greatly as to itsphysical dimensions, and can be rendered more or less electron-rich.

As a result, very high selectivity for one ionic species in the presenceof one or more other ions can be achieved. For example, the Experimentalsection, Section 10, infra, illustrates the measurement of potassiumconcentration in solutions which contain relatively high concentrationsof sodium. Thus, it is not only the structure and chromogenicity of thepresent compound which render it unique, but also, and perhaps moreimportantly, its adaptability to being fashioned to suit the intendedion of interest, thereby achieving heretofore unattainable selectivityfor one type of ion in solution in the presence of another, even whenthe concentration of the latter far outstrips the former.

Accordingly, each branch of the bicyclic system (I) is variable, both inphysical dimension, degree of electron-richness or electron deficiency,and in the nature of substituent groups. For example, by varying thenumber of the groups CR₂ OCR₂ in each of the chains in which it occurs,the electron density affecting the cavity can be designed to suit boththe charge of the ion to be detected as well as its ionic radius andother physical dimensions.

6.1 Cationic Adaptability

The chromogenic cryptahemispherands of the present invention can be madeadaptable to the detection of cations. The bridgehead nitrogen atoms areuncharged, and their unshared electron pairs are available toparticipate with other electron-rich atoms in the molecule in renderingit an electron-rich environment conducive to receiving and complexingwith a cation. Moreover, because of the unique steric configurationalaspects of the cavity of the molecule, contributed in part by thearomatic chain of the bicyclic structure, the molecule can virtually"lock in" the entrapped ion, thereby dramatically increasing theassociation constant, K_(a), of the complex. Other ions in the testsample which might be attracted by the election-rich cavity are eithertoo large to penetrate it or too small to be held by the cavity geometryand structure, thus leading in both cases to a very low K_(a) forcompeting ions in comparison to that of the ion for which the bicyclicionophore has been tailored.

6.2 The Chromogenic Moiety

Compound I includes as part of its structure a particular kind ofchemically configured moiety, Q, which is capable of changing itsphysico-chemical characteristics when a complex is formed by an ion andcompound (I). That is to say, if the target ion, i.e., the ion for whichthe structure of (I) has been tailored to selectively accept to form anionophore/ion complex, is present in a test sample, whether or not otherions are present, a detectable change in those physico-chemicalproperties takes place. This capability of Q to exhibit such a responseto complexation contributes greatly to the usefulness of (I) in assayingthe analyte, or target, ion.

Whereas the concept of the chromogenic moiety Q is very broad, includingwithin its scope a plethora of known and yet-to-be-discovered chemicaland physical configurations, nevertheless several common threads existamong them, and are possessed by each. As the structure (I) indicates, Qmust be divalent. Thus it is capable of bonding within the aromaticchain of the bicyclic structure through at least two covalent bonds.Secondly, as mentioned above, it must be capable of taking on differentattributes when (I) is complexed with an ion than when (I) is in itsuncomplexed state.

As presently contemplated, it is preferred that Q have the genericstructure II: ##STR4## in which R is as defined supra and G is achemical moiety which, when attached as depicted, acts by itself or inconcert with the rest of the depicted structure (II) to form adetectable response to a complexed ion. Thus the concept of G is broad,and includes, but is not limited to, such chemical moieties as ##STR5##as well as any other moiety, known or to be discovered, which imparts toQ the desired detectability. Especially preferred for use as group G are2,4,6-trinitroanilino; 2,6-dinitro-4-trifluoroethylanilino;2,4-dinitro-6-trifluromethylanilino; 4-nitroanilino; 4-nitrophenylazo;4-nitrostyryl; and 4-benzoquinonmonoimino. It has been found thatcompound (I) is especially useful when Q has the structure ##STR6##

6.3 Presently Preferred Embodiment

Of the myriad compounds embodied by the present disclosure, one whichhas been found especially selective in the determination of K⁺, such asin blood, serum, and urine, is the compound having the structure of FIG.5 derived from compound (I) wherein:

Q is compound (III);

R is hydrogen;

R' is CH₃ ;

R" is CH₃ ;

a and b are 1;

m and n are 1; and

x and y are 2.

The chromogenic cryptahemispherand of FIG. 5 has been found to exhibitunusually high selectivity for potassium ion, even in solutions havingmany times higher concentrations of other monovalent cations such assodium. Moreover, compositions useful in such analyses can be formulatedand used at a relatively mild pH, such as in the range of about 5-9,preferably between 6 and 8. Yet another advantage of the preferredembodiment is that it is capable of functioning in an essentiallyaqueous environment, without the attendant need of a separatehydrophobic phase. Thus, the latter disadvantageous requirements ofprior art ionophoric test systems have been eliminated by the advent ofthe present invention.

7. THE TEST COMPOSITION

The discovery of the compounds previously described prompted furtherresearch which led to the formulation of a composition which, whenprepared as an aqueous solution, was found useful for detecting thepresence of certain ions, such as potassium, sodium, lithium, andothers. Such composition includes, in addition to compound (I), thepresence of a buffer to provide a pH environment of about 5 to about 9.Preferably the buffer provides a pH of about 6 to 8. In addition, thecomposition may contain manufacturing excipients, stabilizers,surfactants and other inert ingredients, all of which are easily withinthe ken of one skilled in the art, or which could be routinelydetermined at the bench without the need for undue experimentation.

In use the test sample is merely contacted with the composition and thedetectable response is observed. In the case of the compound of FIG. 5,it has been found convenient to assess the response as light absorbedsuch as at 500 nanometers (nm). To a small amount of an aqueous testsample is added a relatively large volume of a solution of the compoundof FIG. 5 at a pH of about 6-8. The mixture is put into a cuvette andobserved spectrophometrically at about 500 nm. Experiments using variedknown potassium concentrations yield a dose/response curve enablingclear correlation between change in absorbance corresponding to variouspotassium concentrations in the millimolar range.

8. THE TEST DEVICE

As the discovery of chromogenic compound (I) led to a composition usefulfor detecting certain ions, so the composition led to a test device,thereby still further extending the utility of the basic discoverycomprising the overall invention. Thus, by incorporating a suitablecarrier matrix with the composition, a test device is obtained whichfacilitates ion assay yet further.

Such a device lends itself to dry storage when not in use, thus enablinglong shelf-life, and can be pressed into service immediately simply bycontacting it with a small portion of the test sample, be it blood,serum, urine or other aqueous solution to be assayed. It can take onsuch formats as a dip-and-read strip for urine or a test slide for usewith an automatic blood analyzer, or can from a multilayer structuresuch as is described in U.S. Pat. Nos. 3,992,158 and 4,292,272.

8.1 The Carrier Matrix

It is desirable that the carrier matrix comprise a porous or wettablematerial. Thus, in a single layer format the carrier matrix can beformed from materials such as paper, cardboard, porous polymers, polymerfiber and natural felts, and other suitable materials. Especiallypreferred as carrier matrix materials are filter paper, and porous highdensity polyethylene. In a multilayer analytical element format, thebuffer can be stored in an upper layer and the chromogeniccryptahemispherand in a lower layer in a superposed laminar fashion. Thematrices for these layers can be formed from materials such as gelatin,water soluble or water swellable polymers, and other suitable materials.In addition to these two layers, a spreading layer, a reflecting layerand a support material can be incorporated to form an integralanalytical element.

8.2 Making the Test Device

The device is prepared by incorporating the carrier matrix with the testcomposition and, if desired, providing the dried matrix with a support.

Thus the composition is applied to the matrix by innoculating thesurface of the matrix or by dipping it into a solution of thecomposition. The thus-impregnated matrix can then be dried at roomtemperature or at elevated temperatures, provided the temperature is notso high as to deleteriously affect the composition.

The dried, impregnated carrier matrix can then be mounted, if desired,on a suitable support such as a circumferential frame which leaves thematrix exposed in the middle; or the matrix can be mounted at one end ofa plastic strip, the other end serving as a convenient handle.

Another way of making the test device, for the analysis of potassium forinstance, can comprise the treatment of a porous high densitypolyethylene matrix with a surfactant to render it wettable, theimpregnation of a reagent mixture containing the compound of FIG. 5, abinder and a buffer, and the drying of the reagent mixture on the porousmatrix.

In use the test sample is contacted with the surface of the test deviceand the detectable response is measured at 580 nm or other wavelength ona reflectometer. Experiments using varied known potassium concentrationsyield a dose/response curve enabling clear correlation between changesin percent reflectance and potassium concentration in the millimollarrange.

9. USE OF THE INVENTION

The present invention can be adapted for use in carrying out a myriad ofion assays, both manually and on automated systems, which assays areapplicable to a broad field. Not only is clinical chemistry part of thatfield, but also chemical research, chemical process control, and qualityassurance are a few of the many possible applications of thistechnology. The composition and test device are well suited for use inclinical testing of body fluids such as blood, blood serum and urine,since in this work a large number of repetitive tests are frequentlyconducted, and test results are often needed soon after the test sampleis taken from the patient.

The test composition and device are used by contacting with the testsample, and observing a detectable response. In a typical analyticalprocedure, a portion of test sample is placed on the test device for asufficient period of time (such as several minutes). If desired, excesssample may be removed, such as by washing in a gentle stream of waterwith subsequent blotting with tissue paper, or washing in a gentlestream of water.

If the ion under analysis is present in the test sample, the complex ofionophore and ion will form, and a detectable response will appear.Where the moiety Q on compound (I) forms or changes color in response tothe complex, such response is observed, either with the naked eye orinstrumentally. Where Q is a fluorophore such as fluoroscein, afluorescence spectrophotometer can be utilized to measure the detectableresponse formed in the test device (here, the appearance of or change influorescence). Other techniques useful in observing a detectableresponse include reflectance spectrophotometry, absorptionspectrophotometry and light transmission measurements.

When the test sample is blood serum, transmission or reflectancetechniques can be used to detect and quantify the presence of anyreaction products, the formation of which serves as the detectableresponse. In this case radiant energy such as ultraviolet, visible orinfrared radiation, is directed onto one surface of the test device andthe output of that energy from the opposite surface is measured.Generally, electromagnetic radiation in the range of from about 200 toabout 900 nm has been found useful for such measurements, although anyradiation permeating the test means and which is capable of signifyingthe occurrence or extent of the response can be used.

Various calibration techniques are applicable as a control for theanalysis. For example, a sample of analyte standard solution can beapplied to a separate test means as a comparison or to permit the use ofdifferential measurements in the analysis.

10. EXPERIMENTAL

A series of experiments was performed to investigate various aspects ofthe present invention. A description of experimental procedures andresults is provided here to assist in the understanding of the basicconcepts as well as to fully and clearly describe preferred embodiments.

10.1 Synthesis of a Preferred Chromogenic Cryptahemispherand

An experiment was performed to synthesize a preferred embodiment ofcompound (I), supra. The chromogenic cryptahemispherand prepared in thisexperiment is referred to in Section 6.3 as the compound of FIG. 5. Thereaction pathway is depicted in FIGS. 1A, 1B and 1C.

Preparation of Compound 2

A suspension of 30 g (0.12 mol) of 1,¹ 34 g (0.2 mol) of benzyl bromide,and 30 g (0.22 mol) of anhydrous K₂ CO₃ in 600 mL of acetone wasrefluxed for 48 hours (h), evaporated under reduced pressure, theresidue was dissolved in CHCl₃ and H₂ O (600 mL of each) and the layerswere separated. The organic extract was dried, concentrated to 50 mL,and added to an Al₂ O₃ column (400 g) made up in 1:1cyclohexane-benzene. Elution of the column with 3 L of 1:1cyclohexane-benzene gave 32.6 g (80%) of 2 as a colorless oil. The ¹ HNMR spectrum (200 MHz, CDCl₃) gave absorptions at δ5.04 (s, OCH₂, 2H)and 6.8-7.66 (m, ArH, 8H).

Preparation of Compound 3

To a solution of 13.3 g (38.9 mmol) of 2 in 350 mL of THF under Ar at-78° C. was added 85 mL of 1.3M sec-butyllithium (cyclohexane). Afterstirring 8 min, the lithiation solution was cannulated over 8 min into150 g (1.4 mol) of trimethyl borate in 350 mL of THF at -78° C. Themixture was stirred 30 min at -78° C., warmed to 0° C. over 1 h, dilutedwith 500 mL of 2 N hydrochloric acid, and stirred 1 h at 25° C. Ether(0.8 L) was added, the mixture was stirred 8 h at 25° C., and the layerswere separated. The aqueous layer was extracted with fresh ether (2×200mL). Evaporation of the ether extracts (no drying) at 25°/30 mm gave 7.8g (91%) of 3 as a moist oil which was stored at 5° C. and used withoutfurther purification. The 1H NMR spectrum [200 MHz, (CD₃)₂ CO]absorptions at δ5.04 (s, ArCH₂, 2H) and 7.14-7.86 (m, ArH, 8H).

Preparation of Compound 5

To a solution of 120 g (0.33 mol) of 4 (iodination of commerciallyavailable p-cresol via literature preparation)² in 1 L THF at 0° C.under Ar was added 35 g (0.73 mol) of NaH (50% in mineral oil). Afterthe vigorous reaction subsided, the cooling bath was removed, 76 g (0.6mol) of dimethyl sulfate was added, and the mixture refluxed 6 h. Themixture was cooled to 25° C. and CH30H was cautiously added to decomposeexcess dimethyl sulfate. Ethyl ether and 10% aqueous NaCl were added(600 mL of each), the layers were separated, and the organic layer wasdried, evaporated and the residue was dissolved in 100 mL ofcyclohexane. The solution was passed through a column containing 1 kgAl₂ O₃ made up in petroleum ether. Elution of the column with CH₂ Cl₂-petroleum ether mixtures (2-10% CH₂ Cl₂) gave 5 as a colorless oil(lit. mp 25° C.)³ in 82% yield (102 g). The ¹ H NMR spectrum (200 MHz,CDCl₃) gave absorptions at δ2.24 (s, ArCH₃, 3H), 3.82 (s, OCH₃, 3H), and7.57 (s, ArH, 2H).

Preparation of Compound 6

A solution of 100 g (0.27 mol) of 5 in 1 L of ether under Ar was cooledto -78° C. A 110 mL portion of 2.5 M BuLi was added over 5 min and theresulting mixture stirred 10 min at -78° C. Carbon dioxide gas wasvigorously bubbled through the suspension for 20 min, and the cold bathwas allowed to warm to 25° C. over 10 h. The suspension was diluted with600 mL of 1 N aqueous NaOH, and the layers were separated. The aqueouslayer was acidified with 6 N HCl and the white solid collected and driedat 25° C. under vacuum to give 50 g (64%) of crude 6. The 1H NMRspectrum [200 MHz, (CD₃)₂ CO] gave absorptions at δ2.33 (s, ArCH₃, 3H),3.85 (s, OCH₃, 3H), 7.64 (d, ArH, 1H), and 7.86 (d, ArH, 1H).

Preparation of Compound 7

To a solution of 50 g (0.17 mol) of 6 in 400 mL of ether at 10° C. wasadded excess CH₂ N₂ (in ether). After stirring 10 minutes at 25° C., theexcess CH₂ N₂ was decomposed with acetic acid and the ether evaporated.The residue was dissolved in 40 mL of CH₂ Cl₂ and flash chromatographedon 300 g of silica gel made up in CH₂ Cl₂. Elution of the column withCH₂ Cl₂ gave 47 g (90%) of 7 as a colorless oil. The ¹ H NMR spectrum(200 MHz, CDCl₃) gave absorptions at δ2.30 (s, ArCH₃, 3H), 3.85 (s,OCH₃, 3H), 3.92 (s, OCH₃, 3H), 7.59 (d, ArH, 1H), and 7.78 (d, ArH, 1H).

Preparation of Compound 8

To a mixture of 7.8 g (35 mmol) of 3 and 27 g (88 mmol) of 7 in 200 mLof benzene and 50 mL of ethanol under Ar was added 100 mL of 2M aqueousNa₂ CO₃. To this vigorously stirred two-phase mixture was added 1.2 g (1mmol) of tetrakis (triphenylphosphine)palladium and the mixture wasrefluxed 48 h (Note: 100 mg of fresh catalyst was added after 24 hreflux).⁴ The layers were separated and the organic layer was dried,evaporated and dissolved in 40 mL of CH₂ Cl₂. The mixture was separatedby flash chromatography on silica gel (250 g) made up in CH₂ Cl₂.Elution of the column with ether-CH₂ Cl₂ mixtures (1 and 2% ether, 2 Lof each) gave 12.8 g (67%) of 8 as a colorless foam. The ¹ H NMRspectrum (200 MHz, CDCl₃) showed absorptions at δ2.32 (s, ArCH₃, 6H),3.57 (s, OCH₃, 6H) 3.93 (s, OCH₃, 6H), 4.33 (s, OCH₂, 2H), and 6.60-7.61(m, ArH, 12H).

Preparation of Compound 9

A suspension of 2 g (2 mmol) of 10% palladium on carbon and 11.1 g (20.6mmol) of 8 in 250 mL of ethyl acetate was hydrogenated (3 atm H₂) in aParr shaker for 2 h. After filtration and evaporation of the ethylacetate, the residue was dissolved in 30 mL of CH₂ Cl₂ and purified byflash chromatograph on Si Gel (150 g) made up in CH₂ Cl₂. Elution of thecolumn with 2% ether-98% CH₂ Cl₂ gave 7.1 g (77%) of 9 as a colorlessfoam. The ¹ H NMR spectrum (200 MHz, CDCl₃) showed absorptions at δ2.38(s, ArCH₃, 6H), 3.60 (s, OCH₃, 6H), 3.92 (s, OCH₃, 6H), and 6.97-7.63(m, ArH, 7H).

Preparation of Compound 10

To a stirred solution of 7.1 g (15.8 mmol) of 9 in 500 mL of 1:1 CHCl₃--CH₃ CO₂ H was added 20 mL of 70% HNO₃ over 2 min. After stirring 15min, the solution was diluted with H₂ O (1.2 L) and CHCl₃ (200 mL) andthe organic layer was extracted with H₂ O (3×1.2 L), dried, concentratedto 25 mL and flash chromatographed on Si Gel (200 g) made up in CH₂ Cl₂.Elution of the column with CH₂ Cl₂ (1 L) and 49:1 CH₂ Cl₂ -ET₂ O (3 L)gave 7.1 g (91%) of 10 as a yellow foam. The ¹ H NMR spectrum (200 MHz,CDCl₃) gave absorptions at 2.42 (s, ArCH₃, 6H), 3.65 (s, OCH₃, 6H), 3.94(s, OCH₃, 6H), 7.36 (d, ArH, 2H), 7.72 (d, ArH, 2H), and 8.30 (s, ArH,2H).

Preparation of Compound 11

A mixture of 7.1 g (14.3 mmol) of 20, 20 g (0.16 mol) of dimethylsulfate and 22 g (0.16 mol) of K₂ CO₃ in 500 mL of acetone under Ar wasrefluxed 24 h, evaporated and the residue dissolved in 1 L of 1:1 CHCl₃-H₂ O. The organic layer was dried, concentrated to 25 mL and flashchromatographed on 200 g of Si Gel made up in CH₂ Cl₂. Elution of thecolumn with CH₂ Cl₂ (1 L) and 49:1 CH₂ C12-ether (2 L) gave 6.8 g (93%)of 11 as a colorless foam. The ¹ H NMR spectrum (200 MHz, CDCl₃) gaveabsorptions at δ2.39 (s, ArCH₃, 6H), 3.30 (s, OCH₃, 3H), 3.60 (s, OCH₃,6H), 3.94 (s, OCH₃, 6H), 7.34 (d, ArH, 2H), 7.68 (d, ArH, 2H), and 8.25(s, ArH, 2H).

Preparation of Compound 12

To a solution of 8 g (15.7 mmol) of 11 in 325 mL of CH OH was added 100mL of H₂ O and then 12 g (0.29 mol) of LiOH·H₂ O. After stirring 14 h at25° C., the mixture was dilute with 400 mL of H₂ O, extracted with CH₂Cl₂ (2×50 mL) and the aqueous layer acidified to pH 1 with concentratedHCl. Extraction of the aqueous suspension with ether (3×300 mL) anddrying for 16 h at 95°/0.01 mm gave 5.6 g (74%) of 12 as an amorphousyellow powder. The ¹ H NMR spectrum [200 MHz, (CD₃)₂ CO] gaveabsorptions at δ2.42 (s, ArCH₃ 6H), 3.37 (s, 3OCH₃, 3H), 3.65 (s, OCH₃,6H), 7.45 (d, ArH, 2H), 7.75 (d, ArH, 2H), and 8.25 (s, ArH, 2H).

Preparation of Compound 13

A suspension of 2.44 g (5 mmol) of 12 in 8 mL (110 mmol) of purifiedthionyl chloride was stirred 2 h at 25° C. under Ar (12 dissolvedafter˜30 min). Dry benzene (30 mL) was added and the solution evaporatedat 40° C./30 mm to remove the excess thionyl chloride. This procedurewas repeated three times. The crude product was dried at 25° C./0.01 mmto give 2.6 g (˜100%) of 13 as a yellow foam and was used withoutfurther purification. The ¹ H NMR spectrum (200 MHz, CDCl₃) gaveabsorptions at δ2.44 (s, ArCH₃, 6H), 3.33 (s, OCH₃, 3H), 3.66 (s, OCH₃,6H), 7.44 (d, ArH, 2H), 8.00 (d, ArH, 2H), and 8.32 (s, ArH, 2H).

Preparation of Compound 15

Compound 13 (2.6 g, 5 mmol) was dissolved in 150 mL of anhydrous benzeneand transferred in 50 mL portions to a 50 mL gas-tight syringe.Similarly, 1.3 g (5 mmol) of 14 (available from Merck Chemicals)together with 1.5 g (15 mmol) of triethylamine was dissolved in 150 mLof anhydrous benzene and transferred to a 50 mL gas-tight syringe. Thesesolutions were added via a syringe pump to an oven-dried 2 liter Mortonflask containing 1200 mL of anhydrous benzene over 2 h with vigorousmechanical stirring under Ar at 12° C. After stirring for 8 h at 12° C.,the suspension was warmed to 25°, filtered to remove triethylaminehydrochloride and evaporated. The residue was dissolved in 40 mL of CH₂Cl₂. Elution of a silica gel column with acetone-dichloromethanemixtures (10-30% of acetone) gave 2.1 g (60%) of 15 as a white solidwhich darkens above 320° C. and melts/decomposes at ˜345° C. The massspectrum (70 eV) showed a molecular ion at m/e 707. The ¹ H NMR spectrum(200 MHz, CDCl₃) showed absorptions at δ2 37 (s, ArCH₃ 6H), 2.85 (s,OCH₃, 3H), 3.41 (s, OCH₃, 6H), 3.05-3.88 (m, NCH₂, OCH₂, 22H), 4.30 (d,NCH₂, 2H), 7.17-7.23 (m, ArH, 4H), and 8.35 (s, ArH, 2H).

Preparation of Compound 16

A suspension of 560 mg (0.79 mmol) of 25 and 1 g of 10% palladium oncharcoal in 200 mL of dimethylformamide was hydrogenated (3 atm H₂) in aParr shaker for 2 h. The catalyst was removed by filtration and thefiltrate diluted with CHCl₃ (500 mL) and H₂ O (1.2 L) and the layerswere separated. The organic layer was extracted with fresh H₂ O (3×1.2L), dried (K₂ CO₃) and evaporated to give 520 mg (97%) of 16 as acolorless foam. The ¹ H NMR spectrum (200 MHz, CDCl₃) showed absorptionsat δ2.32 (s, ArCH₃, 6H), 2.66 (s, OCH₃, 3H), 3.41 (s, OCH₃, 6H),3.06-3.96 (m, NCH₂, OCH₂, 22H), 4.28 (d, NCH₂, 2H), 6.80 (s, ArH, 2H),7.08 (s, ArH, 2H), and 7.13 (s, ArH, 2H).

Preparation of Compound 17

A solution of 490 mg (0.72 mmol) of 16 in 100 mL of THF was heated toreflux under Ar and 2.0 mL (20 mmol) of borane-methyl sulfide was added.The methyl sulfide-THF was slowly distilled from the mixture over 70min. The remaining solution (30 mL) was cooled to 5° C., 5 N aqueousNaCl was cautiously added to decompose excess borane, and THF (30 mL)and 5 N aqueous NaCl (50 mL) were added. The mixture was stirred for 10days at 25° C., the THF was evaporated and the residue was extractedwith CH₂ Cl₂ (2×50 mL). The organic extracts were filtered through phaseseparator paper, concentrated to 5 mL and diluted with 150 mL of CH₃ OH.After adding 0.4 g (4.8 mmol) of NaHCO₃ and 0.2 g (0.81 mmol) of picrylchloride to the CH₃ OH solution and stirring 25 min. at 25° C., themixture was diluted with CH₂ Cl₂ (40 mL) and 100 mL of 1 N aqueous NaCl.The layers were separated, and the organic layer (no drying) was addedto a silica gel column (100 g) made up in 2% CH₃ OH-98% CH₂ Cl₂. Elutionof the column with CH₃ OH--CH₂ Cl₂ mixtures (2-5% CH₃ OH) gave 40 mg(6%) of the 17 KCl complex. The NMR spectrum (200 MHz, CDCl₃) showedabsorptions at δ2.36 (s, ArCH₃,6H), 2.84 (s, OCH₃, 3H), 3.48 (s, OCH₃6H), 2.18-4.10 (m, NCH₂, 24H), 2.67 (d, ArCH₂ N, 2H), 4.20 (d, ArCH₂N,2H), 7.03 (d, ArH, 2H), 7.12 (d, ArH, 2H), 7.17 (s, ArH, 2H) and 9.09(s, ArH, 2H).

Further elution of the column with CH₃ OH-CH₂ Cl₂ mixtures (10-20% CH₃OH) gave 250 mg (38%) of 17 NaCl complex as an orange foam. A Fab massspectrum (m-nitrobenzyl alcohol dispersion) gave a base peak at m/e 883(M+23) corresponding to the M+Na ion and a lower intensity ion at899(M+39, 25% intensity of 883) corresponding to the M+K ion. The ¹ HNMR spectrum of 17·NaCl (200 MHz, CD₂ Cl₂) showed absorptions at δ2.33(s, ArCH₃, 6H), 2.12-4.00 (m, NCH₂ , OCH₂, 24H), 2.95 (d, ArCH₂ N, 2H),4.06 (d, ArCH₂ N, 2H), 4.06 (d, ArCH₂ N, 2H), 7.0-7.13 (m, ArH, 6H) and8.85 (s, ArH, 2H).

10.2 A Preferred Aqueous System for Potassium Determination

An experiment was conducted to assess the performance of the presentinvention in the analysis of potassium ion in an aqueous system, in apresently preferred embodiment.

Accordingly, a reagent solution of the invention was prepared bydissolving 15 mg of the compound of FIG. 5, as its sodium salt, in 1.65mL diethylene glycol monoethyl ether. To this was added 48 mL of 0.1MHEPES buffer⁵ (pH=7.3), followed by 0.17 mL of Brij-35⁶ solution (30%w/v) in distilled water, and the mixture thoroughly stirred.

A spectrophotometric automated instrument known as the RA-1000®systemavailable from Technicon Instruments Corporation was used to assay thesamples. The following instrument parameters were used:

Sample volume: 5.5 μl

Reagent volume: 385.0 μl

Optical filter: 500 nm

Temperature: 37° C.

Delay: 5 min.

Assay Type: end point

Calibration Factor: 1.0

The spectrophotometric data obtained from this procedure is shown inFIG. 2, wherein potassium concentration is plotted against the change inlight absorbance (ΔAbsorbance) at 500 nm. It can be seen that a lineardose-response curve, having a slope conducive to easy differentiationbetween absorbence levels, is obtained.

Results

The preferred aqueous system of the present invention yielded a lineardose/response curve with a slope enabling easy point differentiationusing photometric methods (Δabsorption at 500 nm).

10.3 Use of Preferred Aqueous System for Potassium Determination inSerum

An experiment was conducted to compare the present invention with anart-established procedure for measuring potassium in serum.

A series of random serum samples containing a broad range of potassiumconcentration was obtained. These were analyzed on a RA-1000® system asin 10.2, supra, and also by the RA-1000® ISE mode. The instrumentparameters were the same as those in Section 10.2 for the lightabsorbance mode.

Results

The comparative data is shown in FIG. 3, and shows excellent correlationbetween the method of the present invention and the standard ISE methodfor potassium concentrations in the range of 1-10 mM.

10.4 Effect of pH on Potassium/Sodium Selectivity in a Liquid/LiquidPartitioning System

An experiment was conducted to study the selectivity of a compound ofthe present invention for potassium ion in the presence of sodium ion,where the pH of the aqueous phase was varied within an extraction systemcontaining an immiscible organic solvent.

Two sets of 6 test samples were prepared, one containing potassiumchloride, the other sodium chloride. Stock buffer solutions wereprepared at pH 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0. To a 2.0 ml aliquot ofeach solution was added 0.1 mL of 0.1 M KCl to form the first set ofsamples. The procedure was repeated except 0.1 mL of 0.1 M NaCl was usedinstead of KCl to form the second set of samples. To each sample wasthen added 2 mL of 7×10⁻⁵ M of the compound of FIG. 5 in methylenechloride. Each sample was then thoroughly agitated on a Vortex mixer for1-2, minutes. The samples were set aside briefly to allow phaseseparation, and the absorbance of the CH₂ Cl₂ phase was then measured at300-700 nm on a Beckman DU-8 spectrophotometer. A blank sample was runto provide a control. The blank was prepared as indicated above exceptthat deionized water was used instead of KCl or NaCl solution.

Results

The results are shown in Table 1 in terms of change in light absorbancefrom the control data at 450 nm (ΔA). The data shows that significantresponse to both sodium and potassium occurred at pH levels in the rangeof 7.0 to 10.0, indicating poor discrimination between K⁺ and Na⁺,whereas at pH levels below 7.0, selectivity ratios of from 17.1 to 5.4were obtained. This increase in ion selectivity with lowering pH wasunexpected.

                  TABLE 1                                                         ______________________________________                                        Effect of pH on Sodium and Potassium Response                                 Utilizing Extraction in CH.sub.2 Cl.sub.2 (Δ A at 450 nm)               pH                                                                            5.0        6.0      7.0    8.0    9.0  10.0                                   ______________________________________                                        Na.sup.+                                                                              0.009  0.054    0.509                                                                              0.475  0.493                                                                              0.400                                K.sup.+ 0.154  0.293    0.682                                                                              0.503  0.586                                                                              0.493                                ______________________________________                                    

10.5 Effect of pH and a Water-Miscible Organic Solvent onPotassium/Sodium Selectivity in an Aqueous System

An experiment was conducted to show the effects of (a) pH and (b) theconcentration of a water-soluble organic solvent, on a preferred aqueoussystem of the present invention. Accordingly, solutions of KCl and NaClwere prepared in water at varying pH levels using standard buffers andwith varying amounts of dioxane added.

Aqueous 0.1M buffer solutions were prepared, to yield solutions at a pHof 6;0, 6.6, 7.0, 8.0, and 9.0. To each of these was added an amount ofthe compound of FIG. 5 in dioxane to assure a final concentration of 0.1mM of the compound of FIG. 5. The volume of dioxane was varied toachieve concentrations of 1%, 25% and 50% by volume of dioxane. Thusthree sets of reagent solutions were prepared, all being 0.1 mM of thecompound of FIG. 5. Each set comprised the 5 pH levels, but each setvaried from one another in dioxane percentage.

To 2.0 mL of each sample of reagent was added 0.1 mL of 1.0 M NaCl orKCl in water in an optical cuvette. Following mixing, light absorbancewas measured on a Beckman DU-8 spectrophotometer at 300-700 nm. The datais shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Effect of pH and Dioxane on Sodium and Potassium                              Responses (Δ A at 450 nm) to the                                        Compound of FIG. 5 in Aqueous Medium                                                   pH                                                                   Dioxane    6.0      6.6    7.0    8.0  9.0                                    ______________________________________                                        1%      Na.sup.+                                                                             0.005    0.000                                                                              0.002  0.026                                                                              0.000                                        K.sup.+                                                                              0.003    0.414                                                                              0.512  0.098                                                                              0.006                                25%     Na.sup.+                                                                             0.002    0.001                                                                              0.000  0.016                                                                              0.086                                        K.sup.+                                                                              0.014    0.035                                                                              0.018  0.352                                                                              0.175                                50%     Na.sup.+                                                                             0.002    0.000                                                                              0.013  0.039                                                                              0.072                                        K.sup.+                                                                              0.032    0.012                                                                              0.007  0.061                                                                              0.005                                ______________________________________                                    

Results

In one set of data, that utilizing pH 7 solutions (0.1M HEPES buffer)with 1% dioxane, no response to sodium was detected, whereas aconsiderable response to potassium occurred. Accordingly, the presentinvention exhibits an enormously high selectivity ratio for potassiumover sodium at neutral pH with negligible organic solvent present (SeeFIG. 3). Such unexpected selectivity in chromogenic ionophores isheretofore unreported.

The overall data in Table 2 shows that as the organic solvent portion ofthe reagent was increased, both selectivity and sensitivity forpotassium over sodium decreased. This phenomenon is contrary to resultsdescribed in previously published works, where other ionophores,particularly crown ethers and cryptands, exhibited increased sensitivityand selectivity with increasing porportions of organic reagents. Thepresent invention exhibits the reverse phenomenon.

Moreover, sensitivity and specificity appears inversely proportional topH, whereas the above--mentioned previous results with other ionophoresgenerally exhibited the opposite tendency.

10.6 A Model Test Device

An experiment was performed to prepare a test device of the presentinvention capable of detecting the presence of potassium, whereby acarrier matrix of high density polyethylene (HDPE) was incorporated withthe compound of FIG. 5.

Porous disks having 1/2 inch diameters, a thickness of 1/32 inch, and a35 um pore size were obtained from Porex Technologies, Inc., Fairburn,GA. These were pretreated by saturating with a 1% w/v solution ofSurfynol 104 nonionic detergent (Air Products, Inc.) in chloroform anddrying. The disks were then each treated with 30 uL of reagent solution.The stock reagent solution comprised a mixture of 0.9 mL distilledwater, 0.1 mL diethylenglycolmonoethyl ether, 5 mg of the compound ofFIG. 5 and 40 mg polyvinylpyrrolidone. The treatment compriseddepositing on one side of each disk a 30 uL aliquot of stock reagentsolution, which permeated the entire disk, and allowing the disks to dryat room temperature for five hours with subsequent storage in adessicator charged with anhydrous calcium sulfate for 2 hours.

The disks were tested by innoculation with 25 uL of analytical specimensof 0.2 M MES buffer⁷ at pH 6, containing concentrations of 1.0 mM, 2.0mM, 3.0 mM, 5.0 mM and 7.5 mM, respectively, in potassium.

Following 2 minutes incubation with analytical specimen, the disks wereobserved at 580 nm for reflectance data using an Infra-Alyzer®(Technicon Instruments Corporation) modified for use in the visibleportion of the electromagnetic spectrum.

Results

Reflectance measurements R were transformed into K/S values utilizingthe well-known equation of Kubelka and Munk ##EQU2## K/S values areplotted against potassium concentration in FIG. 4. The curvedemonstrates that the test device possesses ideal sensitivity forpotassium in the clinical range.

10.7 Test Device for Detecting Potassium in Serum

A porous high density polyethylene substrate, 35 um pore size and 1/32inch thick was die cut into 1/2 inch diameter disks. These disks wererendered hydrophic by treatment with 1% Surfynol 104 (Air Products andChemicals, Wayne Pennsylvania) in chloroform and drying. A thirtymicroliter reagent aliquot containing 0.4M imidazole-phosphoric acidbuffer at pH 5.8, 6% polyvinylpyrrolidone (MW 40,000), 0.02% Brij-35(ICI Americas Inc., Wilmington, Del.), 10% 2-ethoxyethoxy ethanol, and 9millimolar compound of FIG. 5 was deposited to each porous high densitypolyethylene disk. These reagent impregnated disks were allowed to dryat ambient conditions for four hours before being used for potassiummeasurement.

To test the response of these dry test devices to various concentrationsof potassium ions in serum samples, thirty microliters serum test samplewas applied to each disk and incubated at room temperature for fiveminutes. The color changes were recorded on a reflectometer at 580 nm.The change in percent reflectance (%R) is indicative of a colorimetricresponse. The result is summarized in Table 3.

To evaluate the accuracy of the determination of potassium ion in humanserum samples, the same samples obtained from a hospital were analyzedfor potassium using a flame photometer and compared with the potassiumvalues obtained using the dry test device method. Correlation databetween the two methods are as follows: slope, 0.995; intercept, 0.063;correlation coefficient, r, 0.991.

                  TABLE 3                                                         ______________________________________                                        Response of the compound of FIG. 5 to potassium ions in                       serum on "dry" test devices.                                                  [K.sup.+ ] mM                                                                              Response (% R)                                                   ______________________________________                                        2            26.5                                                             4            21.7                                                             6            18.6                                                             8            17.5                                                             10           16.5                                                             ______________________________________                                    

10.8 A Preferred Aqueous System for Sodium (Rate) Measurement

An experiment was conducted to assess the performance of one example ofthe present invention in the analysis of sodium ion in an essentiallyaqueous reaction system.

Accordingly, a reagent solution was prepared by dissolving 18 mg of thecompound of FIG. 6 as its lithium bromide complex, in 1.65 mLdiethyleneglycol monoethyl ether. To this was added 48 ml of 0.2M HEPESbuffer pH 7.3 followed by 0.13 mL of TRITON X-100 and the mixturethoroughly stirred.

The RA-1000® from Technicon Instruments was used to assay samples bydiscerning the change in absorbance of individual sample and reagentmixtures over a period of nine minutes and using the followinginstrument parameters:

Sample volume: 4.0 μl

Reagent volume: 395 μl

Optical filter: 500 nm

Temperature: 37° C.

Delay: 15 sec.

Incubation: 9 min.

Calibration Factor: 1.0

Printer Format: 3

Assay Type: rate

The spectrophotometric data obtained from this procedure with aqueoussodium chloride calibrants is linear over the clinically significantrange of 80mM to 200mM sodium in human serum. Specific values for thecalibration curve are: slope, 0.0023 Δabsorbance units per mM in sodiumconcentration, intercept -3.025; correlation coefficient, r, 0.9996.

To evaluate the accuracy of the determination of sodium ion in humanserum, samples obtained from a hospital were analyzed for sodium usingthe RA-1000® Ion Selective Electrode and compared with the sodium valuesobtained using the spectrophometric method. Correlation data between thetwo methods are as follows: slope, 1.059; intercept, -8.82; correlationcoefficient, r, 0.9852.

10.9 A Preferred Aqueous System for Sodium (End Point) Measurement

The performance of another example of the present invention was assessedfor the assay of sodium in serum samples using an essentially aqueousreaction system. Accordingly, 33 mg of the compound of FIG. 7 as itslithium bromide complex, were dissolved in 1.65 mL of diethyleneglycolmonoethyl ether. To this was added 48 ml of 0.2M HEPES buffer pH 8.1followed by 0.085 mL of Brij .35 30% (w/v) and the mixture thoroughlystirred.

The RA-1000® from Technicon Instruments was used to assay samples bydiscerning the change in absorbance of the reaction mixture after sampleis added. The following parameters were used on the instrument:

Sample volume: 2 μL

Reagent volume: 400μL

Optical filter: 550 nm

Temperature: 37° C.

Delay: 9 mins.

Calibration factor: 1.0

Printed format: 3

Assay Type: end point

The spectrophotometric data obtained from this procedure showed a linearrelationship between the change in absorbance of the reaction mixtureand the logarithm of the sodium ion concentration in the sample over theclinically significant range of 80 mM to 200 mM, with a sensitivity ofabout 0.002 absorbance unit per mM change in sodium concentration.

Comparison of serum samples assayed by this spectrophotometric methodand by the RA-1000® Selective Electrode method were also made.Correlation data between the two methods are as follows: slope, 0.963;intercept, 6.22; correlation coefficient, r, 0.9946.

10.10 Test Device for Detecting Sodium Ions

A porous high density polyethylene substrate, 35 un pore size and 1/32inch thick was die cut into 1/2 inch diameter disks. These disks wererendered hydrophilic by treatment with 1% W/V surfynol 104 (Air Productsand Chemicals, Wayne, Pa.) in chloroform and drying. A thirtymicroliters reagent aliquot containing 0.2M imidazole-phosphoric acidbuffer pH 7.5, 0.2% W/V Triton X-100 (Rohn and Haas Co., Philadelphia,Pa.), 10% W/V 2-ethoxyethoxyethanol, 7% W/V polyvinylpyrrolidone (MW40,000), and 3 millimolar compound of FIG. 6 was deposited to eachporous high density polyethylene disk. These reagent impregnated diskswere allowed to dry at ambient conditions for four hours before beingused for sodium measurement.

To test the response of these dry test devices to various concentrationsof sodium ions in aqueous medium, thirty microliters aqueous testsolution was applied to each disk and incubated at 37° C. for sixminutes. The color changes were recorded on a reflectometer at 560 nm.The change in percent reflectance (%R) is indicative of a colorimetricresponse. The result is summarized in Table 4. The data clearly showthat the compound of FIG. 6 can be used to detect sodium ions in a drytest device.

                  TABLE 4                                                         ______________________________________                                        Response of the compound of FIG. 6 to sodium ions in                          aqueous medium on dry test devices.                                           [Na.sup.+ ] mM                                                                              Response (% R)                                                                             K/S                                                ______________________________________                                        0             21.4         1.443                                              2             18.2         1.838                                              4             17.6         1.929                                              6             16.9         2.043                                              8             16.5         2.113                                              10            15.2         2.365                                              15            14.0         2.641                                              ______________________________________                                    

What is claimed is:
 1. A method for selectively determining the presence of a test cation in a test sample, comprising the steps of:(a) contacting said test sample with a compound which complexes selectively to the test cation and being of the formula: ##STR7## wherein: R, the same or different, is hydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl; R', the same or different, is lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl; R", the same or different, is hydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or unsubstituted aryl; Q is a chromogenic moiety capable of providing a detectable response upon the complexation of said compound with said test cation; a, b, m and n, the same or different, are 1 to 3; x is 1 to 4; and y is 1 to 4; and y is 1 to 4; and (b) measuring said detectable response.
 2. The method of claim 1 wherein said chromogenic moiety Q of said compound has the structure: ##STR8## wherein G is 2,4,6e-trinitroanilino; 2,6-dinitro-4-trifluoromethylanilino; 2,4-dinitro-6-trifluoromethylanilino; 4-nitro-anilino; 2,4-dinitrophenylazo; 4-nitrophenylazo; 4-nitrostyryl; and 4-benzoquinonmonoimino.
 3. The method of claim 2 wherein when R is not hydrogen, said chromogenic moiety Q has the structure: ##STR9## wherein: Y, the same or different, is an electron withdrawing group such as CN, NO₂, CF₃ or COOR wherein R is hydrogen, lower alkyl, lower alkylidene, lower alkenyl, allyl or aryl.
 4. The method of claim 3, wherein G is 2,4,6-trinitroanilino, 2,6-dinitro-4-trifluoromethylanilino, 2,4-dinitro-6-trifluoromethylanilino or 4-nitroanilino.
 5. The method of claims 1 to 4 wherein said compound is incorporated into a solid carrier member with a buffer capable of providing a pH in the range of about 5-9, and contacting said test sample with said solid carrier member.
 6. A test device for determining the presence of a test cation in an aqueous test sample pursuant to the method of one of claims 1-4, said device comprising a solid carrier member, the compound of one of claims 1-4 incorporated into said solid carrier member and a buffer incorporated into said carrier member capable of providing a pH in the range of about 5-9. 